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Abstract:

The present invention relates to a material for induction of hard tissue
regeneration, comprising platelet-rich plasma and gelatin β-TCP
sponge, which promotes angiogenesis, osteogenesis, chondrogenesis and the
like.

Claims:

2. The material according to claim 1, wherein the hard tissue is a bone
or a cartilage tissue.

3. The material according to claim 1, which promotes osteogenesis or
chondrogenesis.

4. The material according to claim 1, which promotes angiogenesis.

5. The material according to claim 1, wherein the platelet-rich plasma is
derived from a subject to be applied with a material for induction of
hard tissue regeneration.

6. The material according to claim 1, wherein the gelatin β-TCP
sponge has a pore size of 10-500 μm.

7. The material according to claim 1, wherein the gelatin in the gelatin
β-TCP sponge is crosslinked.

8. The material according to claim 1, wherein the gelatin β-TCP
sponge is prepared by crosslinking and freeze-drying a composition
comprising β-TCP and gelatin.

9. The material according to claim 1, wherein the gelatin constituting
the gelatin β-TCP sponge is an acidic gelatin.

10. The material according to claim 9, wherein the hard tissue is a bone
or a cartilage tissue.

11. The material according to claim 9, which promotes osteogenesis or
chondrogenesis.

12. The material according to claim 9, which promotes angiogenesis.

13. The material according to claim 9, wherein the platelet-rich plasma
is derived from a subject to be applied with a material for induction of
hard tissue regeneration.

14. The material according to claim 9, wherein the gelatin β-TCP
sponge has a pore size of 10-500 μm.

15. The material according to claim 9, wherein the gelatin in the gelatin
β-TCP sponge is crosslinked.

16. The material according to claim 9, wherein the gelatin β-TCP
sponge is prepared by crosslinking and freeze-drying a composition
comprising β-TCP and gelatin.

17. The material according to claim 9, wherein the gelatin β-TCP
sponge is crosslinked and has a pore size of 10-500 μm.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a material for induction of hard
tissue (e.g., bone or cartilage and the like) regeneration, comprising
platelet-rich plasma and gelatin β-TCP sponge.

BACKGROUND ART

[0002] Conventionally, autogenous bone transplantation is performed for
synostosis of bone fracture part, and reconstruction of bone defective
part after excision of benign osteoncus and the like, by harvesting bone
from own healthy part and transplanting the bone in the bone defective
part. However, due to the invasion on a healthy part, complications are
developed in a high rate of about 30%, such as infection of and residual
pain in the bone donor site, nerve damage near the bone donor site and
the like. In a case where the area of synostosis and bone defect are
large, a mass of autogenous bone is necessary, the defective autogenous
bone donor site may sometimes cause a clinical problem.

[0003] To avoid autogenous bone transplantation and compensate for the
shortage of autogenous bone amount, an artificial bone using calcium
phosphate and β-tricalcium phosphate (β-TCP) has been used.
However, since artificial bone itself does not have an osteoinductive
capacity, its applicable site is limited to a comparatively small bone
defect under good bone formation environment, and since ossification
potential is inferior as compared to autogenous bone, it takes some time
to obtain dynamic strength, thus problematically requiring load
restriction and rest for a long time after operation. With such
background, bone formation techniques with osteoinductive capacity
applicable to extensive synostosis and bone defect have been widely
studied.

[0004] In recent years, a technique for culturing own bone marrow
mesenchymal stem cells and transplanting the cells into a bone defective
part in combination with an artificial bone has been developed and
clinical application has been reported (non-patent document 1). However,
it lacks broad utility, since various problems are accumulated in that
specialized facilities such as clean room where cell culture can be
performed (cell processing center) are necessary, which makes it
practically difficult for municipal hospitals to employ the technique,
mesenchymal cell with multilineage potential has a risk of canceration,
differentiation induction is not necessarily perfect and the like.

[0005] Moreover, as a bone formation treatment using a growth factor, bone
morphogenetic protein-2 (BMP-2) produced by genetic recombination was
approved for use in vertebral body fusion by Food and Drug Administration
in Europe and the United States in 1997, and is clinically applied in US.
In recent years, however, its effectiveness and safety are questioned.
For example, a report has documented that, in lumbar interbody fusion
using BMP-2, bone resorption of fixed vertebral body and transplanted
bone occurred at high frequency to drastically decrease the synostosis
rate. In addition, there are reports on inflammatory reaction in lumbar
vertebral peripheral tissues by MRI useful for the evaluation of soft
tissues, and severe complications such as difficult breathing due to
pharyngeal edema, dysphagia and abnormal swelling of cervical part
observed in a case of cervical fixing (non-patent documents 2 and 3). The
cause thereof is considered to be edematous change in the soft tissue and
vital organs resulting from an antigen-antibody reaction induced by
administration of a large amount of artificially-produced growth factor
which diffused from the administration site to the peripheral tissues. In
addition, since BMP-2 is expensive, it adds several hundreds of thousands
of yen to the operation cost, producing a financial burden on patients.

[0006] Platelet-rich plasma (also referred to as PRP) is a concentrated
platelet plasma obtained by removing red blood cell from peripheral blood
by centrifugal separation at a low speed. PRP contains a large amount of
growth factors such as platelet-derived growth factor (PDGF) contained in
platelet, transforming growth factor β (TGF-β), fibroblast
growth factor (FGF), insulin-like growth factor (IGF) and the like, and
is known to show effects such as angiogenesis, osteogenesis, promotion of
wound healing and the like by a synergistic action of these. It has also
been confirmed that a combined use of PRP and an appropriate drug
delivery system material such as gelatin hydrogel and the like exhibits
osteogenetic activity in long bone and skull bone defect models (patent
document 1, non-patent documents 4 and 5).

[0007] However, the effect of PRP alone is not sufficient to afford
sufficient ossification in large bone defect, anatomically unfused bones,
and spinal fusion to joint together spinal bones, and a scaffold having a
three-dimensional structure that promotes differentiation and induction
of osteoblasts is absolutely necessary.

[0015] A problem of the present invention is to provide a novel material
for the regeneration of a hard tissue such as bone or cartilage and the
like, which provides an effect of sustained release of a platelet-rich
plasma-derived growth factor, and good scaffolding function for tissue
regeneration, and is superior in safety and practicality.

Means of Solving the Problems

[0016] To achieve the above-mentioned problem, the inventors have prepared
a material for induction of hard tissue regeneration, comprising a
gelatin β-TCP sponge obtained by processing gelatin, which is
superior as a material for a drug delivery system of growth factor, and
β-TCP granules, which are ceramics having bioabsorbability and
biodegradability, into a sponge-like form, and platelet-rich plasma (PRP)
purified from patient's own blood, which is contained in the sponge. They
have verified that the material strikingly enhances ossification in a rat
spinal fusion model.

[0017] The present invention has been made based on the above-mentioned
finding and provides a material for induction of hard tissue
regeneration, which contains platelet-rich plasma and gelatin β-TCP
sponge.

[0018] The hard tissue to which the material of the present invention is
applied is a bone or cartilage tissue etc. requiring a scaffold for
regeneration, and also includes those in the dental field such as
alveolar bone and the like.

[0019] The material for induction of hard tissue regeneration of the
present invention promotes osteogenesis and chondrogenesis. In addition,
the material for induction of hard tissue regeneration of the present
invention also promotes angiogenesis.

[0020] The material for induction of hard tissue regeneration of the
present invention is preferably used for regeneration of hard tissues
such as bone or cartilage and the like requiring a scaffold, since the
gelatin β-TCP sponge functions as a good cell scaffold for tissue
regeneration. However, the application site thereof is not limited to a
hard tissue, and it may be a soft tissue in contact with a hard tissue
such as bone or cartilage and the like. To be specific, the material
induces regeneration of sinew and ligament by its angiogenesis promoting
action even in the case of a transplant surgery of a soft tissue such as
sinew, ligament etc. in contact with bone or cartilage. Therefore, such
use for inducing regeneration of a soft tissue in contact with a hard
tissue is also encompassed in the present invention.

[0021] The platelet-rich plasma to be used in the present invention is
preferably derived from a subject to be applied with a material for
induction of hard tissue regeneration. The subject to be applied with the
material for induction of hard tissue regeneration of the present
invention is not limited to human and includes mammal as a whole.

[0022] The gelatin β-TCP sponge to be used in the present invention
preferably has a pore size of about 10-500 μm, and gelatin in the
gelatin β-TCP sponge is preferably crosslinked. The aforementioned
crosslinking is formed by subjecting a composition containing β-TCP
and gelatin to crosslinking and freeze-drying. The order of crosslinking
and freeze-drying may be any, and a composition after crosslinking may be
freeze-dried or a freeze-dried composition may be subjected to
crosslinking.

Effect of the Invention

[0023] PRP used in the present invention is advantageously free of ethical
problems associated with treatments and the risk associated with the use
of blood preparations such as virus infection, immuno-incompatibility and
the like, since it can be easily harvested from the blood of the subject
to be applied with a material for induction of hard tissue regeneration.
Moreover, the gelatin β-TCP sponge to be used in the present
invention releases a component contained in PRP, such as growth factor
and the like, in a sustained manner to promote osteogenesis,
chondrogenesis and angiogenesis, as well as functions as a good cell
scaffold for tissue regeneration, thereby enabling remarkable bone
formation.

[0029] The present specification encompasses the content described in the
specification of the priority base application No. 2009-241041.

DESCRIPTION OF EMBODIMENTS

[0030] The present invention relates to a material for induction of hard
tissue regeneration, which contains platelet-rich plasma (PRP) and
gelatin β-TCP sponge.

1. Platelet-Rich Plasma (PRP)

[0031] The "platelet-rich plasma (PRP)" of the present invention is a
concentrated platelet plasma obtained by removing red blood cell from
peripheral blood by centrifugal separation at a low speed. PRP contains a
large amount of growth factors such as platelet-derived growth factor
(PDGF) contained in platelet, transforming growth factor β
(TGF-β), stem cell derived factor (SDF)-1, insulin-like growth
factor (IGF) and the like, and is known to show effects such as
angiogenesis, osteogenesis, promotion of wound healing and the like by a
synergistic action of these.

[0032] As for the centrifugation conditions for the preparation of PRP,
the rotation number and time for efficient removal of blood cell
components can be appropriately determined according to the kind of the
centrifugal separator to be used. If necessary, platelets may be
concentrated by centrifuging again after removal of the blood cell
components. Alternatively, PRP can also be prepared by a blood separator
using a membrane.

[0033] PRP to be used in the present invention is preferably derived from
a subject's own blood, who is in need of administration of the material
for induction of hard tissue regeneration of the present invention.
However, when the subject has severe anemia or an underlying disease, due
to which a blood sample is difficult to obtain, platelet-rich plasma
purified from blood of other person may be used.

2. Gelatin β-TCP Sponge

[0034] The gelatin β-TCP sponge used in the present invention is a
porous sponge-like structure consisting of gelatin and β-TCP
granule.

2.1 Gelatin

[0035] The gelatin used in the present invention can be obtained by
denaturing collagen obtainable from any parts such as skin, bone, sinew
and the like of the body of various animal species such as cow, pig, fish
and the like, or a substance used as collagen by various treatments such
as alkali hydrolysis, acid hydrolysis, enzymatic hydrolysis and the like.
While the properties of gelatin vary depending on the material and
treatment method to be used, any gelatin having such properties can be
utilized as a gelatin β-TCP sponge material in the present
invention.

[0036] Examples of the measure showing the properties of gelatin include
isoelectric point, molecular weight, zeta potential and the like. For
example, commercially available gelatin includes type A gelatin
manufactured by Sigma Ltd. and gelatin manufactured by Wako Pure Chemical
Industries, Ltd., and the zeta potential in an aqueous solution is as
follows:

[0037] type A gelatin manufactured by Sigma Ltd.: about 0 to about 5 mV

The zeta potential is a measure showing the electrostatic charge level of
a substance (gelatin).

[0039] Particularly preferable gelatin to be used in the present invention
has the following properties: acidic gelatin obtained from collagen by an
alkali hydrolysis treatment,

molecular weight about 10-about 200,000 daltons by SDS-PAGE under
non-reduced conditions, and zeta potential in an aqueous solution about
-15 to about -20 mV.

[0040] Preferably, moreover, acidic gelatin prepared from bovine
bone-derived type I collagen by an alkali treatment can be used, which
can be obtained from Nitta Gelatin Inc. with an isoelectric point (IEP)
5.0. While a basic gelatin prepared by an acid treatment can also be
obtained from Nitta Gelatin Inc. as sample IEP 9.0, the zeta potential is
greatly different as shown below.

[0043] β-TCP (tricalcium phosphate) to be used in the present
invention is biodegradable ceramics, which is conventionally used widely
as an artificial bone material. The compressive strength of porous
β-TCP is about 3M Pa, which is weaker than biological bone (about 7M
Pa for cancellous bone) but sufficiently strong for clinical use.
β-TCP is gradually decomposed in the body and release calcium ion
and phosphoric acid ion to realize an environment that facilitates
synthesis of hydroxyapatite, which is a bone constituent component, by
osteoblast. That is, β-TCP not only functions as a carrier for a
drug delivery system or a scaffold for ossification, but also positively
promotes osteogenesis, chondrogenesis, angiogenesis and the like. Thus,
β-TCP provides a good scaffold material which increases the dynamic
strength of a gelatin sponge obtained by mixing with gelatin, as well as
positively promotes osteogenesis, chondrogenesis, angiogenesis and the
like. The size, porosity, pore size and the like of β-TCP to be used
in the present invention may be any, and any kind of β-TCP granule
can be used. For example, commercially available β-TCP-100
(pulverized product): manufactured by Taihei Chemical Industrial Co.,
Ltd., OSferion(registered trade mark): manufactured by OLYMPUS
CORPORATION and the like can be used.

2.3 Structure of Gelatin β-TCP Sponge

[0044] The ratio of gelatin and β-TCP in the gelatin β-TCP
sponge of the present invention is 10:1-1:10, preferably 5:1-1:5, in dry
weight.

[0045] The gelatin β-TCP sponge of the present invention is like a
sponge having many ultrafine pores having an average pore size of 10-500
μm. Having a structure including many ultrafine pores, the sponge
permits easy entry of the surrounding cells thereinto upon
transplantation, and can function as a scaffold material for tissue
regeneration. Furthermore, sufficient nutrition and oxygen can be
supplied to the adhered cells, thus enabling normal cell growth and
differentiation.

[0046] The lower limit of the average pore size of the ultrafine pores in
the gelatin β-TCP sponge of the present invention is 10 μm, and
the upper limit thereof is 500 μm. When it is less than 10 μm,
since cells cannot enter into a support for tissue engineering, the cell
adhesiveness becomes extremely inferior and adhered cells cannot expand
three-dimensionally. When it exceeds 500 μm, since the cell density
becomes low, tissues and organs cannot regenerate. A preferable lower
limit is 50 μm, and a preferable upper limit is 200 μm.

[0047] In the gelatin β-TCP sponge of the present invention, gelatin
is crosslinked. An index for evaluating the crosslinking level (degree of
crosslinking) is water content. The water content is a weight percentage
of water in a swollen gelatin β-TCP sponge relative to the weight of
the sponge. When the water content is high, the degree of crosslinking of
the gelatin β-TCP sponge decreases, and the sponge is easily
degraded. That is, the enzymatic degradability of the gelatin β-TCP
sponge in the body changes depending on the water content, and the water
content affects sustained release (gradual release) of PRP.

[0048] The gelatin β-TOP sponge of the present invention preferably
has a water content of 90%-99.8%. When the water content is less than
90%, flexibility suitable for transplantation is sometimes impaired,
biodegradation may take a long time after transplantation into the body,
and a physiologically active substance may remain in a support without
being released in a sustained manner. When it exceeds 99.8%, it may occur
that a support cannot maintain its strength in a culture medium or
buffer, and a physiologically active substance is gradually released only
for a short period of 1 to 3 days. More preferable lower limit is 95% and
more preferable upper limit is 98%.

2.4 Production Method of Gelatin β-Top Sponge

[0049] The gelatin β-TOP sponge to be used in the present invention
is obtained by subjecting a composition containing gelatin and β-TOP
to crosslinking and freeze-drying.

[0050] To be specific, β-TCP is added to gelatin. Then, gelatin is
crosslinked. The crosslinking method is not particularly limited and, for
example, vacuum thermal dehydration method, dry heating method, γ
ray irradiation method, UV irradiation method, electron beam irradiation
method, X ray irradiation method, a method using a crosslinking agent and
the like can be mentioned. Of these, a method using a crosslinking agent
is preferable since, as mentioned below, even gelatin once formed like a
sponge can be crosslinked with the same degree of crosslinking even to
the inside of the sponge.

[0051] The crosslinking agent to be used is not particularly limited and,
for example, glutaraldehyde, water-soluble carbodiimide such as EDC and
the like, and condensing agent that produces a chemical bond between
propylene oxide, diepoxy compound, hydroxyl group, carboxyl group, amino
group, thiol group, imidazole group and the like (ethylene glycol
diglycidyl ether, polyethylene glycol diglycidyl ether,
polyglycerolpolyglycidyl ether, glycerolpolyglycidyl ether,
hexamethylenediisocyanate etc.) can be used. Preferred is glutaraldehyde.

[0052] Crosslinking may be performed before freeze-drying or after
freeze-drying. Specifically, for example, a sponge-like formed product is
obtained by foaming an aqueous gelatin solution containing β-TCP by
vigorously stirring the solution in a homogenizer and the like,
performing a crosslinking reaction, casting the reaction mixture in a
mold, and freezing and further freeze-drying the same, or foaming an
aqueous gelatin solution containing β-TCP by vigorously stirring the
solution in a homogenizer and the like, casting the reaction mixture in a
mold, freezing and further freeze-drying to give a sponge-like formed
product, and immersing the sponge-like formed product in a glutaraldehyde
solution having a suitable concentration for a given time to perform
crosslinking (see JP-A-2005-211477).

[0053] To discontinue a crosslinking reaction by glutaraldehyde, which is
reactive with an amino group, for example, a low molecular substance
having an amino group such as ethanolamine, glycine and the like only
needs to be brought into contact therewith, or an aqueous solution having
pH 2.5 or less may be added. To completely remove the crosslinking agent
and low molecular substance used for the reaction, the obtained gelatin
β-TCP sponge may be washed with distilled water, ethanol,
2-propanol, acetone and the like, and freeze-dried again.

[0054] A sponge structure critical as a scaffold material can be formed by
foaming by stirring and freeze-dry step. That is, innumerable ice
crystals grown by the freezing step become pores by freeze-drying, and a
porous sponge structure having a desired pore ratio and a pore size can
be formed.

[0055] Moreover, to avoid swelling of a sponge-like structure in the
initial stage of a treatment with a crosslinking agent such as
glutaraldehyde and the like, a thermal crosslinking treatment and the
like may be applied before treatment. In addition, when crosslinking by a
crosslinking agent is performed, toxicity is preferably removed by
treating the reaction terminal. For example, after a glutaraldehyde
treatment, toxicity can be removed by deactivating the reaction terminal
by washing with aqueous glycine solution and the like.

[0056] The gelatin β-TCP sponge may be formed in any shape and can be
formed in, for example, cylindrical form, prismatic form, sheet form,
disc form, spherical form, particle and the like. The cylindrical,
prismatic, sheet and disc forms are often generally used as embedded
pieces, or can also be used as particles after pulverization. In
addition, spherical form and particle form can also be administered in an
injection.

3. Material for Induction of Hard Tissue Regeneration of the Present
Invention

[0058] The "hard tissue" in the present invention means a tissue having a
hard intercellular substance including bone, cartilage and teeth. The
material for induction of hard tissue regeneration of the present
invention is used as a material for induction of hard tissue regeneration
since it contains PRP having a regeneration promoting effect on
osteogenesis, chondrogenesis, angiogenesis and the like, and gelatin
β-TCP sponge superior as a scaffold for tissue regeneration.
Particularly, it is preferably used as a material for induction of
regeneration of defective bone or cartilage tissue.

[0059] As mentioned above, while the material for induction of hard tissue
regeneration of the present invention is preferably used as a material
for induction of regeneration of defective bone or cartilage tissue
requiring a cell scaffold, the applicable region thereof is not limited
to a hard tissue, and may be a soft tissue in contact with a hard tissue
such as bone or cartilage and the like. To be specific, the material
induces regeneration of sinew and ligament by the angiogenesis promoting
action of PRP even in the case of a transplant surgery of a soft tissue
such as sinew, ligament etc. in contact with bone or cartilage.
Therefore, such use for inducing regeneration of a soft tissue in contact
with a hard tissue is also encompassed in the present invention.

[0060] The material for induction of hard tissue regeneration of the
present invention can be widely used the fields of medicine and
veterinary medicine including orthopedic and dental field, and the
subject of application thereof is not limited to human, and includes
mammals in general.

[0061] The material for induction of hard tissue regeneration of the
present invention is prepared by adding PRP to the above-mentioned
gelatin β-TCP sponge. PRP-containing gelatin β-TCP sponge can
be obtained by, for example, adding dropwise PRP to the above-mentioned
freeze-dried gelatin β-TCP sponge, or immersing gelatin β-TCP
sponge in PRP to allow impregnation of the sponge with PRP. This
immersing operation generally ends in 15 min-1 hr at 4-37° C.,
preferably 15-30 min at 4-25° C., during which the sponge is
swollen with PRP, and a growth factor and the like contained in PRP
interact with gelatin molecule in the sponge, whereby the growth factor
and the like form a complex with the gelatin molecule, thus fixing PRP in
the gelatin β-TCP sponge by a physical interaction. It is considered
that not only the static interaction between them but also other
interactions such as hydrophobic bond, hydrogen bond and the like greatly
contribute to the formation of a complex between PRP and gelatin
molecule.

[0062] The weight ratio of PRP to a gelatin β-TCP sponge (dry weight)
is preferably within the range of about 1-fold to about 10000-fold. More
preferably, PRP relative to a gelatin β-TCP sponge shows a weight
ratio of about 2-fold to about 5000-fold, more preferably about 10-fold
to about 1000-fold.

[0063] The material for induction of hard tissue regeneration of the
present invention is directly embedded (applied to) in an affected part
and can be devised to have an appropriate dosage form suitable for each
use. That is, the gelatin β-TCP sponge may be formed to have a
desired form such as cylindrical, prismatic, sheet, disc, spherical,
particular form, and the like according to the application site thereof.

[0064] The dose of PRP in the material for induction of hard tissue
regeneration of the present invention can be appropriately adjusted
according to the severity of disease, and age, body weight and the like
of the subject. In the case of a human, the dose for an adult patient is
generally selected from the range of about 0.1-about 500 ml, preferably
about 1-about 50 ml, and this can be administrated into an affected part
or the vicinity thereof. When the effect obtained by a single
administration is not sufficient, the administration can be performed
multiple times.

[0065] The material for induction of hard tissue regeneration of the
present invention may contain, where necessary, other appropriate
medicaments and pharmacologically acceptable carriers. Examples of such
medicament and carrier include a medicament that promotes angiogenesis
and bone formation, an activator of osteoblast or an inhibitor of
osteoclast, a scaffold carrier that promotes cell growth and
differentiation, a combination thereof, and the like.

4. Effect of the Material for Induction of Hard Tissue Regeneration of the
Present Invention

4.1 Effect of PRP Sustained Release

[0066] Since the material for induction of hard tissue regeneration of the
present invention shows both a PRP sustained-release effect and
stabilization effect, a small amount thereof can exert the functions of
various growth factors and the like contained in PRP for a long time.
Consequently, the angiogenesis promoting function and osteogenesis,
chondrogenesis ability of the growth factors and the like can be
effectively exerted in the topically administered site.

[0067] The mechanism of sustained release is based on the fact that
various growth factors and the like contained in PRP are physically fixed
on gelatin β-TCP in the sponge. The present inventors have
heretofore tried sustained release of growth factor, cytokine, monokine,
lymphokine, other physiologically active substances and the like by using
a bioabsorbable polymer hydrogel, and succeeded in a sustained release of
a growth factor and the like having a physiological activity unachievable
by other materials, and control of the duration of the sustained release.
In the present invention, the growth factor and the like contained in PRP
are considered to be released in a sustained manner from gelatin
β-TCP due to a similar mechanism. A growth factor etc. fixed on
gelatin β-TCP are not released from the sponge. Along with the
degradation of gelatin β-TCP sponge in the body, gelatin β-TCP
molecule becomes water soluble, which causes release of the growth factor
and the like fixed on the gelatin β-TCP molecule. That is,
degradation of gelatin β-TCP sponge enables control of sustained
release of the growth factor and the like. The degradability of gelatin
β-TCP sponge can be altered by controlling the degree of
crosslinking during production of the gelatin β-TCP sponge. In
addition, since various growth factors and the like contained in PRP
interact with gelatin β-TCP, their stability in the body, for
example, resistance to enzymatic degradation and the like, is improved.
Moreover, since gelatin β-TCP sponge disappears along with the
sustained release of growth factor and the like, the bone formation
process due to the growth and differentiation of cells is not physically
inhibited.

[0068] The PRP to be contained in the material for induction of hard
tissue regeneration of the present invention contains various growth
factors and the like, and provides various effects such as angiogenesis
promoting effect, osteogenesis promoting effect, chondrogenesis promoting
effect, wound healing promoting effect, skin ulcer treatment effect and
the like (as mentioned above).

[0069] As shown in the below-mentioned Examples, the inventors have
confirmed that the material for induction of hard tissue regeneration of
the present invention has a remarkable bone forming action. Ossification
is reported to involve angiogenesis and, combined with the contribution
of osteogenesis and chondrogenesis promoting effect but also various
effects of PRP such as angiogenesis promoting effect etc., tissue
regeneration is promoted in the defective region and good bone formation
is achieved.

4.3 Effect as Scaffold Material

[0070] The material for induction of hard tissue regeneration of the
present invention comprises a crosslinked gelatin β-TCP sponge as a
main component, and a substrate having many ultrafine pores having a
given pore size. Thus, the material permits easy entry of cells and can
function as a scaffold material for regeneration. In addition, since PRP
component is electrostatically bound to the surface of the substrate and
released in a sustained manner along with the decomposition of the
substrate, it can act on the cell for a long time, thus strikingly
increasing the ossification effect. The rate of sustained release can be
controlled by adjusting the degree of crosslinking of gelatin. That is,
the material for induction of hard tissue regeneration of the present
invention can simultaneously play two roles of a scaffold material for
regeneration and a regeneration promoter.

EXAMPLES

[0071] The present invention is explained in more detail in the following
by referring to Examples, which are not to be construed as limitative.

Examples

1. Preparation of Platelet-Rich Plasma (PRP)

[0072] SD rat (male, 8-week-old, 240-29.0 g, SHIMIZU Laboratory Supplies
Co., Ltd., Kyoto) was placed under general anesthesia by intraperitoneal
injection of pentobarbital (250 μL). The chest was opened and blood (5
CC) was drawn from the heart using a 21G needle. The blood was
transferred to a 15 cc centrifugal tube containing ACD-A solution (W/V %:
citric acid 0.85, trisodium citrate 2.32, glucose 2.59) (2 CC) in
advance. The centrifugal tube was centrifuged in a centrifuge (KN-70,
manufactured by KUBOTA CORPORATION) at 1500 rpm for 10 min. After
centrifugation, a transparent serum portion in the upper layer was
suctioned with a 14G Surflo needle and transferred to another centrifugal
tube and centrifuged at 3000 rpm for 10 min. The serum portion in the
upper layer was suctioned with a 14G Surflo needle leaving 200 and the
remaining solution was stirred to give platelet-rich plasma (PRP) (200
μL). A gelatin β-TCP sponge cut into a 2 mm cube
(β-TCP-reinforced gelatin sponge MedGel (registered trade mark)
Scaffold (MedGEL CO., LTD.), total 20 mg) was immersed in the obtained
PRP (200 μL) and left standing at 4° C. overnight. As a
control, a gelatin β-TCP sponge in the same amount and shape was
immersed in phosphate buffer saline (hereinafter PBS, 200 μL) and left
standing at 4° C. overnight.

[0074] The paraspinal muscle was longitudinally incised for 25 mm at 5 mm
right and left from the midline, and the transverse processes of the
fourth and fifth lumbar vertebrae (hereinafter transverse processes 4, 5)
were exposed on the both sides. Using a steel bar with a 0.5 mm diameter
tip (OSADASUCCESS-40M, OS-40MV, manufactured by OSADA, INC.), the dorsal
cortical bone of the transverse processes was drilled until confirmation
of the bleeding from the bone marrow. The materials shown below were
transplanted in between the transverse processes 4, 5, and the incised
paraspinal muscles and skin were sutured with a 3-0 nylon thread
(manufactured by company). The materials to be transplanted were 1: PRP
impregnated gelatin β-TCP sponge (hereinafter PRP sponge), 2: PBS
(phosphate buffer saline) impregnated gelatin β-TCP sponge (MedGel
(registered trade mark) Scaffold (MedGEL CO., LTD.): hereinafter, PBS
sponge), 3: PRP alone, 4: autologous iliac bone, and no-transplantation
group, total 5 groups. In the PRP alone model of 3, only 200 μL of PRP
was sprayed in between the transverse processes. As the self-ilium of 4,
the tip of ilium on the same side as the scheduled transplantation site
was taken in the size of in 5×2×2 mm. FIG. 1 schematically
shows transplantation in between transverse processes 4, 5 in rat spinal
posterolateral fusion model.

[0075] 3. Evaluation of Ossification and Evaluation of Mechanical Strength

[0076] At 8 weeks after the operation, the rat was euthanized by high dose
administration of pentobarbital, and the lumbar vertebrae were isolated.
Using μCT (microfocus 2D/3D X-ray CT apparatus, ScanXmate-E090S40,
manufactured by Comscantecno Co., Ltd.), sagittal reconstitution images
of the transverse processes 4, 5 were formed, and the presence or absence
of concrescence between the transverse processes was evaluated. In
addition, based on the obtained images and using an image analysis
software (FanCT ver1.3 manufactured by Comscantecno Co., Ltd.), only the
part between the transverse processes was extracted and the bone mass was
measured. Thereafter, a three-point bending test of the lumbar vertebrae
was carried out by a three-point bending test machine (TOKYO TESTING
MACHINE, LSC-200/30-2). The lumbar vertebrae were set with the front
facing upward on a fixture with a span of 40 mm, and the 4/5 lumbar disc
front was pressed at speed of 10 mm/min using a cylindrical fixture
(diameter 5 mm). The load at the time point when the displaced distance
was 10 mm was compared between groups. Then, the tissue was fixed with 4%
para-formaldehyde at 4° C. for 1 week. The tissue was decalcified
with 0.5M EDTA (ethylenediaminetetraacetic acid) solution for 2 weeks and
immersed in 20% sucrose for 2 days. A frozen tissue section (thickness 12
μm) between transverse processes 4, 5 was prepared and stained with
hematoxylin-eosin and safranine O (cartilage substrate was stained red).

4. Results

[0077]FIG. 2 shows the results of the sagittal section reconstitution
images of the left and right transverse processes 4, 5. The PRP sponge
model and autologous iliac bone model were found to show concrescence
tendency between the left and right transverse processes. In addition,
the PRP sponge model showed marked increase of anteroposterior diameter
of transverse processes, as compared to the autologous iliac bone model.
The PBS sponge, single PRP and no-transplantation model did not show an
increase between transverse processes and a synostosis tendency was not
found.

[0078]FIG. 3 shows the bone mass between transverse processes 4, 5. The
PRP sponge model showed a remarkable increase of the bone mass as
compared to the other models.

[0079] FIG. 4 shows the results of the three-point bending test. The PRP
sponge model showed 59.6-fold strength on average as compared to the
other groups.

[0080] FIG. 5 shows tissue images between transverse processes 4, 5 of PRP
sponge model. Between transverse processes, bone substrate and hyaline
cartilage components are mixed in the images, and an intracartilaginous
ossification-like phenomenon was observed. By safranine O-staining, a
part around hyaline cartilage showed a red stained image, verifying it is
a cartilage substrate. For comparison, tissue images (8 weeks
post-operation) between transverse processes 4, 5 of no-transplantation
group is shown. As shown in FIG. 5, ligament component is observed
between transverse processes, but bone and cartilage components are not
observed.

5. Conclusion

[0081] From the above results, it has been verified that a combination of
PRP and gelatin β-TCP sponge achieves an extremely high bone or
cartilage formation ability exceeding that of autogenous bone
transplantation, and the mechanical strength at the synostosis part is
markedly potentiated.

[0083] The acid-treated gelatin was dissolved in distilled water to give 3
wt % aqueous gelatin solution. To the obtained aqueous gelatin solution
(60 mL) was added β-TCP (1.8 g) and 0.16 wt % aqueous glutaraldehyde
solution (0.4 mL) was added, and the mixture was stirred in a homogenizer
at 5000 rpm for 3 min to allow foaming. The foamed aqueous gelatin
β-TCP solution was cast in a 12 cm×12 cm mold, left standing
at 4° C. for 12 hr to perform a crosslinking reaction, after which
the solution was frozen at -40° C. and freeze-dried to give a
sponge.

[0084] The obtained sponge was washed 3 times with 0.1N aqueous glycine
solution for 1 hr, further washed with water, and freeze-dried again to
give a gelatin β-TCP sponge.

[0085] All publications, patents and patent applications cited in the
specification are hereby incorporated in their entireties by reference.

INDUSTRIAL APPLICABILITY

[0086] Platelet-rich plasma used in the present invention does not require
special facilities and equipment, since it can be purified by
centrifugation of the own blood of the subject to be applied with a
material for induction of hard tissue regeneration. Therefore, the
present invention can be practiced in many facilities. In addition, since
platelet-rich plasma derived from own blood is used, the possibility of
side effects is extremely low as compared to gene recombinant growth
factor. Furthermore, since gelatin β-TCP sponge comprises only the
materials in clinical use and has high safety for the human body, the
material for induction of hard tissue regeneration of the present
invention is expected to be extremely rapidly subjected to clinical
application. The diffusion of the present invention dramatically
decreases the need for collection of autogenous bone, which has been
generally performed conventionally, and the ossification promoting action
enables early loading and early rehabilitation. Hence, the benefit
thereof is immeasurable.